Description

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/455,727, filed Mar. 17, 2003, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel derivatives of the cyclosporin family of compounds, cyclosporin A in particular, methods for preparing such derivatives, pharmaceutical compositions comprising such derivatives, and methods of using such derivatives for treatment of various diseases.

BACKGROUND OF THE INVENTION

Cyclosporin A, currently marketed as Neoral® and Sandimmune® (Novartis), is the most widely prescribed drug for the prevention of organ transplant rejection. Cyclosporin A has also demonstrated clinical efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis, Crohn's disease, psoriasis, and Type I diabetes and chronic inflammatory diseases like asthma. Test results in many other preclinical studies indicate utility for cyclosporin A in other therapeutic areas.

Widespread use of cyclosporin A for clinical uses other than prevention of organ transplant rejection is limited, however, due to the drugs narrow therapeutic index. Long term toxicity from chronic administration of cyclosporin A is a serious drawback. Negative consequences associated with chronic treatment with cyclosporin A include nephrotoxicity, abnormal liver function, hirsutism, tremor, neurotoxicity, gastrointestinal discomfort, and other adverse effects. Toxicity associated with cyclosporin A usage has been attributed by many experts working in the immunosuppression therapeutic area as mechanism based. Cyclosporin A inhibits the ubiquitous serine/threonine phosphatase called calcineurin. Attempts to separate the immunosuppressive activity from toxicity through structural modification of cyclosporin A have, for the most part, been unsuccessful. Nevertheless, over the past decade, continued investigation into understanding cyclosporin's toxicity has provided other possible explanations that are independent of calcineurin inhibition.

Published results of recent research (Paolini et al., “Cyclosporin A and Free Radical Generation,” Trends in Pharmaceutical Sciences, 22:14-15 (2001); Buetler et al., “Does Cyclosporin A Generate Free Radicals?” Trends in Pharmaceutical Sciences, 21:288-290 (2000)) suggest that cyclosporin A-mediated generation of reactive oxygen intermediates may be linked with the significant side effects that accompany use of this drug. Results of in vitro and in vivo studies indicate that although cyclosporin A is capable of generating reactive oxygen intermediates, the radicals formed are not derived directly from the cyclosporin A molecule, and are unlikely to stem from mitochondria or from cytochrome P450-mediated metabolism of cyclosporin A.

Novel cyclosporin A analogue, ISATX247, is a potent calcineurin inhibitor (Abel et al., “ISATX247: A Novel Calcineurin Inhibitor,” J. Heart Lung Transplant, 20(2):161 (2001); Aspeslet et al., “ISATX247: A Novel Calcineurin Inhibitor,” Transplantation Proceedings, 33(1-2):1048-1051 (2001)) and has demonstrated a reduced toxicity profile relative to cyclosporin A in animal studies. It remains to be shown if this translates into reduced toxicity in human. In PCT International Publication No. WO 99/18120 to Naicker et al., cyclosporin analogues were claimed, where the MeBmt1 ((4R)-4-((E)-2-butenyl)-4, N-dimethyl-L-threonine) amino acid side chain of cyclosporin A has been structurally modified. Some of the most active compounds claimed in this publication have deuterium incorporated in place of one or more hydrogens in the side chain. Incorporation of deuterium is known to slow down metabolism of compounds in vivo, if hydrogen abstraction is the rate limiting step in the metabolism of the drug (Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism,” Trends in Pharmaceutical Sciences, 5:524-527 (1984)), and improve the pharmacokinetic properties of the molecule. Deuterium incorporation in cyclosporin A analogues may also block pathways responsible for generation of reactive oxygen intermediates in a manner not currently understood.

Other studies have implicated the role of transforming growth factor-β (TGF-β) in the nephrotoxicity of cyclosporin A (Khanna et al., “TGF-β: A Link Between Immunosuppression, Nephrotoxicity, and CsA,” Transplantation Proceedings, 30:944-945 (1998)). Cyclosporin A induces expression of TGF-β, collagen and fibronectin genes in the kidney. TGF-β has a host of immunosuppressive effects that parallel the effects of cyclosporin A. However, TGF-β also causes the accumulation of extracellular matrix genes by increasing the expression of collagen and fibronectin, which is the hallmark of fibrosis. Because glomerulosclerosis (which occurs with chronic cyclosporin A use) is associated with an increase of extracellular matrix proteins, cyclosporin A-associated nephrotoxicity may be due to TGF-β induction. Novel analogues of cyclosporin A may have different effects on induction of gene expression of proteins like TGF-β and may demonstrate improved therapeutic index.

Therefore, it would be advantageous to have novel cyclosporin derivatives that are safe and effective for the treatment of a variety of diseases.

The present invention is directed to achieving these objectives.

SUMMARY OF THE INVENTION

The compounds of the present invention are represented by the chemical structure found in Formula (I):

Formula I

wherein A is an amino acid of Formula (II):

where:

R0 is H or CH3;

R1═CHO;

C(═O)OR2;

C(O)NR3R4;

CH═N—Y;

CH(NR5R6)R7;

CH(OR8)R9;

CH(OR10)2;

CH2SR11;

CH(SR12)2;

CR13R14R15;

CH═CHC(═O)Me;

CH2CH2C(═O)Me;

CH═CHCH(OR16)Me;

CH2CH2CH(OR16)Me;

CH═CHCH(NR17R18)Me;

CH2CH2CH(NR17R18)Me;

CH═CHC(═N—Y)Me;

CH2CH2C(═N—Y)Me;

CH═CHC(OR19)2Me;

CH2CH2C(OR19)2Me;

CH═CHC(═CR20R21)Me;

CH2—CH2C(═CR20R21)Me;

CH═CHC(SR22)2Me;

CH2CH2C(SR22)2Me;

CH═CR23R24;

CH2CHR23R24;

CH═CHC(═O)NR25R26;

CH2CH2C(═O)NR25R26;

CH═CHC(═O)OR26;

CH2CH2C(═O)OR26;

CH═CHC(═O)CH2CH2NR27R28;

CH2CH2C(═O)CH2CH2NR27R28;

CH═CHC(═O)CH═CHNR29R30;

CH2CH2C(═O)CH═CHNR29R30;

CH═CH—C(OR31)R32Me;

CH2CH2C(OR31)R32Me;

CH═CHC(═O)CH2C(OH)R33R34; or

CH2CH2C(═O)CH2C(OH)R33R34;

R2 and R26 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

CH2OCH3;

CH2SCH3;

CH2CH2F;

CH2CF3;

CH2CH2CF3;

CH(CF3)2; and

CH2OCH2OC(O)CH3;

R3, R4, R5, R6, R10, R11, R12, R17, R18, R19, R22, R25, R27, R28, R29, and R30 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring; and

(CH2)n-heteroaryl ring;

R3 and R4, R5 and R6, R10, R12, R17 and R18, R19, R22, R25 and R26, R27 and R28, R29 and R30 are together —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and —CH2CH2CH2CH2CH2CH2— that results in the formation of a cyclic moiety that contains the heteroatom or heteroatoms to which they are bound;

R8, R16, and R31 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

alkanoyl;

alkenoyl;

alkynoyl;

aryloyl;

arylalkanoyl;

alkylaminocarbonyl;

arylaminocarbonyl;

arylalkylaminocarbonyl;

alkyloxycarbonyl;

aryloxycarbonyl; and

arylalkyloxycarbonyl;

R7, R9, R13, R14, R15, R20, R21, R23, R24, R32, R33, and R34, are the same or different and independently selected from the group consisting of:

hydrogen;

deuterium;

halogen;

hydroxyl;

nitrile;

substituted and unsubstituted C1-C6-straight alkyl chain;

substituted and unsubstituted C2-C6-straight alkenyl chain;

substituted and unsubstituted C3-C6-branched alkyl chain;

substituted and unsubstituted C4-C6-branched alkenyl chain;

substituted and unsubstituted C2-C6-straight alkynyl chain;

substituted and unsubstituted C4-C6-branched alkynyl chain;

substituted and unsubstituted C4-C6-chain having alkenyl and alkynyl groups;

a modified α-aminobutyric acid, alanine, valine, or norvaline where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline;

and a modified valine or norvaline where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

The present invention also relates to a process of preparation of a product compound of the formula:

The process involves reducing a compound of the formula:

where:

X═OH;

R0═CH3;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline;

and a modified valine or norvaline where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

X═OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

X═OH;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

R23═hydrogen;

deuterium;

halogen;

hydroxyl;

nitrile;

substituted and unsubstituted C1-C6-straight alkyl chain;

substituted and unsubstituted C2-C6-straight alkenyl chain;

substituted and unsubstituted C3-C6-branched alkyl chain;

substituted and unsubstituted C4-C6-branched alkenyl chain;

substituted and unsubstituted C2-C6-straight alkynyl chain;

substituted and unsubstituted C4-C6-branched alkynyl chain;

substituted and unsubstituted C4-C6-chain having alkenyl and alkynyl groups;

substituted and unsubstituted C3-C7-cycloalkyl;

substituted and unsubstituted (CH2)p—(C3-C7-cycloalkyl);

substituted and unsubstituted aryl;

substituted and unsubstituted heteroaryl;

substituted and unsubstituted arylalkyl;

substituted and unsubstituted heteroarylalkyl;

COOH;

COOR2; and

C(O)NR3R4;

R2=hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

CH2OCH3;

CH2SCH3;

CH2CH2F;

CH2CF3;

CH2CH2CF3;

CH(CF3)2; and

CH2OCH2OC(O)CH3;

R3 and R4 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring; and

(CH2)n-heteroaryl ring;

R3 and R4 are together —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and —CH2CH2CH2CH2CH2CH2— that results in the formation of a cyclic moiety that contains the heteroatom or heteroatoms to which they are bound;

n=0, 1, 2, 3 or 4;

p=0, 1, 2, or 3;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

R2=hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

CH2OCH3;

CH2SCH3;

CH2CH2F;

CH2CF3;

CH2CH2CF3;

CH(CF3)2; and

CH2OCH2OC(O)CH3;

n=0, 1, 2 ,3 or 4;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0 ═CH3;

R9=hydrogen;

deuterium;

halogen;

hydroxyl;

nitrile;

substituted and unsubstituted C1-C6-straight alkyl chain;

substituted and unsubstituted C2-C6-straight alkenyl chain;

substituted and unsubstituted C3-C6-branched alkyl chain;

substituted and unsubstituted C4-C6-branched alkenyl chain;

substituted and unsubstituted C2-C6-straight alkynyl chain;

substituted and unsubstituted C4-C6-branched alkynyl chain;

substituted and unsubstituted C4-C6-chain having alkenyl and alkynyl groups;

substituted and unsubstituted C3-C7-cycloalkyl;

substituted and unsubstituted (CH2)p—(C3-C7-cycloalkyl);

substituted and unsubstituted aryl;

substituted and unsubstituted heteroaryl;

substituted and unsubstituted arylalkyl;

substituted and unsubstituted heteroarylalkyl;

COOH;

COOR2; and

C(O)NR3R4;

R2=hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

CH2OCH3;

CH2SCH3;

CH2CH2F;

CH2CF3;

CH2CH2CF3;

CH(CF3)2; and

CH2OCH2OC(O)CH3;

R3 and R4 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring; and

(CH2)n-heteroaryl ring;

R3 and R4 are together —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and —CH2CH2CH2CH2CH2CH2— that results in the formation of a cyclic moiety that contains the heteroatom or heteroatoms to which they are bound;

n=0, 1, 2, 3 or 4;

p=0, 1, 2, or 3;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

R23=hydrogen;

deuterium;

halogen;

hydroxyl;

nitrile;

substituted and unsubstituted C1-C6-straight alkyl chain;

substituted and unsubstituted C2-C6-straight alkenyl chain;

substituted and unsubstituted C3-C6-branched alkyl chain;

substituted and unsubstituted C4-C6-branched alkenyl chain;

substituted and unsubstituted C2-C6-straight alkynyl chain;

substituted and unsubstituted C4-C6-branched alkynyl chain;

substituted and unsubstituted C4-C6-chain having alkenyl and alkynyl groups;

substituted and unsubstituted C3-C7-cycloalkyl;

substituted and unsubstituted (CH2)p—(C3-C7-cycloalkyl);

substituted and unsubstituted aryl;

substituted and unsubstituted heteroaryl;

substituted and unsubstituted arylalkyl;

substituted and unsubstituted heteroarylalkyl;

COOH;

COOR2; and

C(O)NR3R4;

R2=hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

CH2OCH3;

CH2SCH3;

CH2CH2F;

CH2CF3;

CH2CH2CF3;

CH(CF3)2; and

CH2OCH2OC(O)CH3;

R3 and R4 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring; and

(CH2)n-heteroaryl ring;

R3 and R4 are together —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and —CH2CH2CH2CH2CH2CH2— that results in the formation of a cyclic moiety that contains the heteroatom or heteroatoms to which they are bound;

n=0, 1, 2, 3 or 4;

p=0, 1, 2, or 3;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

R23=hydrogen;

deuterium;

halogen;

hydroxyl;

nitrile;

substituted and unsubstituted C1-C6-straight alkyl chain;

substituted and unsubstituted C2-C6-straight alkenyl chain;

substituted and unsubstituted C3-C6-branched alkyl chain;

substituted and unsubstituted C4-C6-branched alkenyl chain;

substituted and unsubstituted C2-C6-straight alkynyl chain;

substituted and unsubstituted C4-C6-branched alkynyl chain;

substituted and unsubstituted C4-C6-chain having alkenyl and alkynyl groups;

substituted and unsubstituted C3-C7-cycloalkyl;

substituted and unsubstituted (CH2)p—(C3-C7-cycloalkyl);

substituted and unsubstituted aryl;

substituted and unsubstituted heteroaryl;

substituted and unsubstituted arylalkyl;

substituted and unsubstituted heteroarylalkyl;

COOH;

COOR2; and

C(O)NR3R4;

R2=hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

CH2OCH3;

CH2SCH3;

CH2CH2F;

CH2CF3;

CH2CH2CF3;

CH(CF3)2; and

CH2OCH2OC(O)CH3;

R3 and R4 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring; and

(CH2)n-heteroaryl ring;

R3 and R4 are together —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and —CH2CH2CH2CH2CH2CH2— that results in the formation of a cyclic moiety that contains the heteroatom or heteroatoms to which they are bound;

n=0, 1, 2, 3 or 4;

p=0, 1, 2, or 3;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

Another aspect of the present invention relates to a process of preparation of a product compound of the formula:

The process involves treating a compound of the formula:

where:

R0═CH3;

R23=hydrogen;

deuterium;

halogen;

hydroxyl;

nitrile;

substituted and unsubstituted C1-C6-straight alkyl chain;

substituted and unsubstituted C2-C6-straight alkenyl chain;

substituted and unsubstituted C3-C6-branched alkyl chain;

substituted and unsubstituted C4-C6-branched alkenyl chain;

substituted and unsubstituted C2-C6-straight alkynyl chain;

substituted and unsubstituted C4-C6-branched alkynyl chain;

substituted and unsubstituted C4-C6-chain having alkenyl and alkynyl groups;

substituted and unsubstituted C3-C7-cycloalkyl;

substituted and unsubstituted (CH2)p—(C3-C7-cycloalkyl);

substituted and unsubstituted aryl;

substituted and unsubstituted heteroaryl;

substituted and unsubstituted arylalkyl;

substituted and unsubstituted heteroarylalkyl;

COOH;

COOR2; and

C(O)NR3R4;

R2=hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

CH2OCH3;

CH2SCH3;

CH2CH2F;

CH2CF3;

CH2CH2CF3;

CH(CF3)2; and

CH2OCH2OC(O)CH3;

R3 and R4 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring; and

(CH2)n-heteroaryl ring;

R3 and R4 are together —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and —CH2CH2CH2CH2CH2CH2— that results in the formation of a cyclic moiety that contains the heteroatom or heteroatoms to which they are bound;

n=0, 1, 2, 3 or 4;

p=0, 1, 2, or 3;

X═OH or OAc;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, where a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine,

under conditions effective to produce the product compound.

The present invention discloses cyclosporin analogues that are safe and effective for the treatment of a variety of diseases. Some of the cyclosporin compounds of the present invention possess potent immunosuppressive activity comparable with known cyclosporins, especially cyclosporin A, as well as other naturally occurring cyclosporins or known synthetic cyclosporin analogues. The cyclosporin analogues of the present invention are produced by a chemical transformation of cyclosporins that possess the MeBmt1 ((4R)-4-((E)-2-butenyl)-4, N-dimethyl-L-threonine) amino acid, including cyclosporin A. Cyclosporin analogues, especially those of CsA, of the present invention include the incorporation of deuterium in the hydrocarbon side chains of the Bmt1 ((4R)-4-((E)-2-butenyl)-4-methyl-L-threonine) amino acid. Many members of the cyclosporin family contain the Bmt1 and MeBmt1 amino acid.

The present invention discloses chemical processes that rely on the use of an oxidative biocatalyst or chemical oxidation conditions to yield derivatives of cyclosporins containing structural modifications to the Bmt1 side chain. The net effect is the conversion of the (E)-2-butenyl terminus of the Bmt1 amino acid to a methyl vinyl ketone moiety. The cyclosporin methyl vinyl ketone derived from cyclosporin A has never been reported before. The methyl vinyl ketone derivatives of other members of the cyclosporin family are also unknown. The cyclosporin A methyl vinyl ketone displays significant immunosuppressive activity in the mixed lymphocyte reaction assay in murine splenocytes and human T-lymphocytes.

The present invention also describes the utility of cyclosporin A methyl vinyl ketone as a synthetic intermediate that is converted to additional cyclosporin derivatives. The methyl vinyl ketone is a versatile functional group that can undergo facile chemical transformation to a wide range of unique cyclosporin analogues with variations at the one amino acid position. While many analogues of cyclosporins, especially cyclosporin A, have been synthesized with modifications at the Bmt1 amino acid since Wenger's landmark total synthesis of CsA (Wenger, “Total Synthesis of Cyclosporin A and Cyclosporin H, Two Fungal Metabolites Isolated from the Species Tolypocladium inflatum GAMS,” Helv. Chim. Acta, 67:502-525 (1984); U.S. Pat. No. 4,396,542 to Wenger, which are hereby incorporated by reference in their entirety), this synthetic route used for CsA analogue preparation is lengthy. As a result, the number of derivatives that have been prepared with Bmt1 structural variations has been limited. Even considering the number of cyclosporin analogues with variations at the Bmt1 amino acid that have already been synthesized and tested, there is still considerable room for further exploration of structure activity relationships on this important part of the cyclosporin molecule. The production of cyclosporin A methyl vinyl ketone gives synthetic access to many novel cyclosporin analogues more efficiently in only a few chemical steps starting from cyclosporin A.

The structure of cyclosporin A, a cycloundecapeptide, and the position numbering for each amino acid in the ring is shown below:

Cyclosporin A is the most prominent member of the cyclosporin family of compounds. Therefore, names and abbreviations for other members of the cyclosporin family are often based on cyclosporin A. For example, cyclosporin B can be designated as [Ala2]Cy A or (Ala2)-Cs A, which indicates that the amino acid alanine is present at the two position instead of the amino acid, α-aminobutyric acid (Abu), that is present at the two position in cyclosporin A.

Important members of the cyclosporin family that have been isolated and characterized are shown in Table 1.

TABLE 1

Cyclosporins

Cy A or CsA = Cyclosporin (Sandimmune ®) = Cyclosporin A

Cy B = [Ala2]Cy A

Cy C = [Thr2]Cy A

Cy D = [Val2]Cy A

Cy E = [Val11]Cy A

Cy F = [Deoxy-MeBmt1]Cy A

Cy G = [Nva2]Cy A

Cy H = [D-MeVal11]Cy A

Cy I = [Val2, Leu10]Cy A

Cy K = [Deoxy-MeBmt1, Val2]Cy A

Cy L = [Bmt1]Cy A

Cy M = [Nva2, Nva5]Cy A

Cy N = [Nva2, Nva10]Cy A

Cy O = [MeLeu1, Nva2]Cy A

Cy P = [Bmt1, Thr2]Cy A

Cy Q = [Val4]Cy A

Cy R = [Leu?, Leu?] Cy A

Cy S = [Nva2, Nva5]Cy A

Cy T = [Leu10]Cy A

Cy U = [Leu6]Cy A

Cy V = [Abu7]Cy A

Cy W = [Thr2, Val11]Cy A

Cy X = [Nva2, Leu9]Cy A

Cy Y = [Nva2, Leu6]Cy A

Cy Z = [N-Methyl-2-amino-octanoic acid1]Cy A

Novel compounds of the present invention are derived from cyclosporins like the ones shown, especially cyclosporin A, where the position one amino acid is:

(a) MeBmt1 (acronym for (4R)-4-((E)-2-butenyl)-4, N-dimethyl-L-threonine; systematic name is (2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoic acid, also called N-Methyl-butenyl-threonine);

The family of cyclosporins also extends to cyclosporin derivatives that do not occur in nature and, here, have been prepared by total synthetic and semi-synthetic methods. Many novel cyclosporin analogues have been prepared by total synthetic methods, allowing for the incorporation of natural or unnatural amino acids for one or more of the eleven amino acids of the cycloundecapeptide. (U.S. Pat. No. 4,396,542 to Wenger; U.S. Pat. No. 4,639,434 to Wenger et al.; European Patent Application No. 34567 to Wenger; Ko et al., “Solid Phase Total Synthesis of Cyclosporin Analogs,” Helv. Chim. Acta, 80(3):695-705 (1997); U.S. Pat. No. 5,948,693 to Rich et al., which are hereby incorporated by reference in their entirety). Cyclosporin analogues prepared by total synthesis belong to the cyclosporin family.

Novel compounds of the present invention are derived from the cyclosporin family that include the unnatural cyclosporins like the ones that were prepared by total synthetic or semi-synthetic methods in the above cited references, but are not limited to the unnatural cyclosporins in these references.

The compounds of the present invention are represented by the chemical structure found in Formula I:

Formula I

where A is an amino acid of Formula II:

where:

R0 is H or CH3;

R1═CHO;

C(═O)OR2;

C(O)NR3R4;

CH═N—Y;

CH(NR5R6)R7;

CH(OR8)R9;

CH(OR10)2;

CH2SR11;

CH(SR12)2;

CR13R14R15;

CH═CHC(═O)Me;

CH2CH2C(═O)Me;

CH═CHCH(OR16)Me;

CH2CH2CH(OR16)Me;

CH═CHCH(NR17R18)Me;

CH2CH2CH(NR17R18)Me;

CH═CHC(═N—Y)Me;

CH2CH2C(═N—Y)Me;

CH═CHC(OR19)2Me;

CH2CH2C(OR19)2Me;

CH═CHC(═CR20R21)Me;

CH2—CH2C(═CR20R21)Me;

CH═CHC(SR22)2Me;

CH2CH2C(SR22)2Me;

CH═CR23R24;

CH2CHR23R24;

CH═CHC(═O)NR25R26;

CH2CH2C(═O)NR25R26;

CH═CHC(═O)OR26;

CH2CH2C(═O)OR26;

CH═CHC(═O)CH2CH2NR27R28;

CH2CH2C(═O)CH2CH2NR27R28;

CH═CHC(═O)CH═CHNR29R30;

CH2CH2C(═O)CH═CHNR29R30;

CH═CH—C(OR31)R32Me;

CH2CH2C(OR31)R32Me;

CH═CHC(═O)CH2C(OH)R33R34; or

CH2CH2C(═O)CH2C(OH)R33R34;

R2 and R26 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

CH2OCH3;

CH2SCH3;

CH2CH2F;

CH2CF3;

CH2CH2CF3;

CH(CF3)2; and

CH2OCH2OC(O)CH3;

R3, R4, R5, R6, R10, R11, R12, R17, R18, R19, R22, R25, R27, R28 , R29, and R30 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring; and

(CH2)n-heteroaryl ring;

R3 and R4, R5 and R6, R10, R12, R17 and R18, R19, R22, R25 and R26, R27 and R28, R29 and R30 are together —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and —CH2CH2CH2CH2CH2CH2— that results in the formation of a cyclic moiety that contains the heteroatom or heteroatoms to which they are bound;

R8, R16, and R31 are the same or different and independently selected from the group consisting of:

hydrogen;

C1-C6-straight alkyl chain;

C3-C6-straight alkenyl chain;

C3-C6-branched alkyl chain;

C4-C6-branched alkenyl chain;

C3-C6-straight alkynyl chain;

C3-C7-cycloalkyl;

CH2—(C3-C7-cycloalkyl);

(CH2)n-aryl ring;

(CH2)n-heteroaryl ring;

alkanoyl;

alkenoyl;

alkynoyl;

aryloyl;

arylalkanoyl;

alkylaminocarbonyl;

arylaminocarbonyl;

arylalkylaminocarbonyl;

alkyloxycarbonyl;

aryloxycarbonyl; and

arylalkyloxycarbonyl;

R7, R9, R13, R14, R15, R20, R21, R23, R24, R32, R33, and R34, are the same or different and independently selected from the group consisting of:

hydrogen;

deuterium;

halogen;

hydroxyl;

nitrile;

substituted and unsubstituted C1-C6-straight alkyl chain;

substituted and unsubstituted C2-C6-straight alkenyl chain;

substituted and unsubstituted C3-C6-branched alkyl chain;

substituted and unsubstituted C4-C6-branched alkenyl chain;

substituted and unsubstituted C2-C6-straight alkynyl chain;

substituted and unsubstituted C4-C6-branched alkynyl chain;

substituted and unsubstituted C4-C6-chain having alkenyl and alkynyl groups;

CO— in Formula II is covalently bound to an α-amino group of B in Formula I to form an amide linkage, and —N—R0 in Formula II is covalently bound to a carboxylic acid of K to form an amide linkage;

B is an amino acid selected from the group consisting of:

α-aminobutyric acid;

alanine;

threonine;

valine;

norvaline; and

a modified α-aminobutyric acid, alanine, valine, or norvaline, wherein a carbon atom in a side chain is substituted with a hydroxyl group;

C is a sarcosine;

D is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

valine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

E is an amino acid selected from the group consisting of:

valine;

norvaline; and

a modified valine or norvaline, wherein a carbon atom in a side chain is substituted with a hydroxyl group;

F is an amino acid selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine;

G is α-aminobutyric acid or alanine;

H is D-alanine;

I and J are independently selected from the group consisting of:

leucine;

N-methyl leucine;

γ-hydroxy-N-methyl leucine; and

γ-hydroxy leucine; and

K is N-methyl valine or valine;

or a pharmaceutically acceptable salt thereof.

The term “C1-C6-straight alkyl chain” as used herein refers to saturated, straight chain hydrocarbon radicals containing between one and six carbon atoms. Examples include methyl, ethyl, propyl, n-butyl, n-pentyl, and n-hexyl.

The term “C3-C6-straight alkenyl chain” as used herein refers to straight chain hydrocarbon radicals containing between three and six carbon atoms with at least one carbon-carbon double bond. The term “C2-C6-straight alkenyl chain” as used herein refers to straight chain hydrocarbon radicals containing between two and six carbon atoms with at least one carbon-carbon double bond. Examples include ethylene, CD=CD2, 2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-butenyl, 2-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, and 4-hexenyl.

The term “C3-C6-branched alkyl chain” as used herein refers to branched chain hydrocarbon radicals containing between three and six carbon atoms. Examples include, but are not limited to, isopropyl, isobutyl, tert-butyl, and neopentyl.

The term “C4-C6-branched alkenyl chain” as used herein refers to branched chain hydrocarbon radicals containing between four and six carbon atoms with at least one carbon-carbon double bond. Examples include 2-methyl-2-propenyl, 3-methyl-2-butenyl, and 4-methyl-3-pentenyl, and the like.

The term “C2-C6-straight alkynyl chain” as used herein refers to straight chain hydrocarbon radicals containing between three and six carbon atoms with a carbon-carbon triple bond. The term “C2-C6-branched alkynyl chain” as used herein refers to straight chain hydrocarbon radicals containing between four and six carbon atoms with a carbon-carbon triple bond. Examples include C≡CH, C≡CCH3, C≡CCH2CH3, C≡CCH2C(CH3)2, CH2C≡CH, CH2C≡CCH3, and the like.

The term “C3-C7-cycloalkyl” as used herein refers to cyclic hydrocarbon radicals between three and seven carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

The term “alkanoyl” used herein refers to a substituted or unsubstituted C1-C6-straight alkyl chain, a C3-C6-branched alkyl chain and a C3-C7-cycloalkyl group covalently bound to a carbonyl group. Examples include acetate, propionate, pivaloate, butyrate, isobutyrate, cyclohexane carboxylate, and the like.

The term “alkenoyl” used herein refers to a substituted or unsubstituted C3-C6-straight alkenyl chain covalently bound to a carbonyl group. Examples include acrylate, crotonate, methacrylate, 2,4-hexadienoate, and the like.

The term “alkynoyl used herein refers to a substituted or unsubstituted C3-C6-straight alkynyl chain covalently bound to a carbonyl group. Examples include propiolate, 2-butynoate, and the like. Substituents on alkanoyl, alkenoyl and alkynoyl chains are in any appropriate position and are independently selected from F, Cl, Br, I, OH, CO2H, CO2Me, CN, C(O)NH2, C(O)NHCH3, OC(O)CH3, OCH3, NH2, NHCH3, N(CH3)2, and NHC(O)CH3.

The term “aryloyl” used herein refers to a substituted or unsubstituted aryl or heteroaryl ring. Examples of arylacyl groups include benzoyl, p-fluorobenzoyl, 2-naphthoyl, nicotinoyl, isonicotinoyl, and the like.

The term “arylalkanoyl” used herein refers to a substituted or unsubstituted aryl or heteroaryl ring covalently bound to an alkyl chain of one, two, three or four carbon atoms whereby one of the carbon atoms of the alkyl chain is covalently attached to a carbonyl group. The alkyl chain is substituted or unsubstituted, straight or branched, saturated or unsaturated. Examples of arylalkylacyl groups include phenylacetoyl, p-fluorophenylacetoyl, 2-phenylpropionoyl, mandeloyl, cinnamoyl, and the like.

The term “alkylaminocarbonyl” used herein refers to a substituted or unsubstituted C1-C6-straight alkyl chain, a C3-C6-branched alkyl chain, and a C3-C7-cycloalkyl group covalently bound to a nitrogen atom that is covalently bound to a carbonyl group. Examples of alkylaminoacyl groups include methylaminocarbonyl, ethylaminocarbonyl, isopropylaminocarbonyl, tert-butylaminocarbonyl, cyclopentylaminocarbonyl, cyclohexylaminocarbonyl, and the like.

The term “arylaminocarbonyl” used herein refers to a substituted or unsubstituted aryl or heteroaryl ring. Examples include phenylaminocarbonyl, (naphth-2-yl)aminocarbonyl, para-methoxyphenylaminocarbonyl, (pyrid-4-yl)aminocarbonyl, (pyrazin-2-yl)aminocarbonyl, and the like.

The term “arylalkylaminocarbonyl” used herein refers to a substituted or unsubstituted aryl or heteroaryl ring covalently bound to an alkyl chain of one, two, three or four carbon atoms whereby one of the carbon atoms of the alkyl chain is covalently bound to an amino group which is covalently bound to a carbonyl group. Examples include benzylaminocarbonyl, phenethylaminocarbonyl, α-methylbenzylaminocarbonyl, pyrid-4-yl methylaminocarbonyl, and the like.

The term “alkyloxycarbonyl” used herein refers to a substituted or unsubstituted C1-C6-straight alkyl chain, a C3-C6-branched alkyl chain, and a C3-C7-cycloalkyl group covalently bound to an oxygen atom that is covalently bound to a carbonyl group. The substituents are in any position and are independently selected from F, Cl, Br, I, OH, CO2H, CO2Me, CN, C(O)NH2, C(O)NHCH3, OC(O)CH3, OCH3, SCH3, NH2, NHCH3, N(CH3)2, and NHC(O)CH3. Examples include methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl (BOC), and the like.

The term “aryloxycarbonyl” used herein refers to a substituted or unsubstituted aryl or heteroaryl ring. Examples include phenyloxycarbonyl, (naphth-2-yl)oxycarbonyl, para-methoxyphenyloxycarbonyl, (pyrid-4-yl)oxycarbonyl, (pyrazin-2-yl)oxycarbonyl, and the like.

The term “arylalkyloxycarbonyl” used herein refers to a substituted or unsubstituted aryl or heteroaryl ring covalently bound to an alkyl chain of one, two, three, or four carbon atoms whereby one of the carbon atoms of the alkyl chain is covalently bound to an oxygen atom which is covalently bound to a carbonyl group. Examples include benzyloxycarbonyl, phenethyloxycarbonyl, α-methylbenzyloxycarbonyl, (pyrid-4-yl)methyl oxycarbonyl, 9-fluorenylmethyl oxycarbonyl (FMOC), and the like.

The cyclosporin nomenclature and numbering systems used herein are those used by Kallen et al., “Cyclosporins: Recent Developments in Biosynthesis, Pharmacology and Biology, and Clinical Applications,” In Biotechnology, second edition, Rehm et al., eds, pp. 535-591 (1997), which is hereby incorporated by reference in its entirety, and are shown below:

Position numbering

Letter in Formula I

Amino acid in cyclosporin A

1

A

N-Methyl-butenyl-threonine

(MeBmt)

2

B

α-Aminobutyric acid (Abu)

3

C

Sarcosine (Sar)

4

D

N-Methyl-leucine (MeLeu)

5

E

Valine (Val)

6

F

N-Methyl-leucine (MeLeu)

7

G

Alanine (Ala)

8

H

(D)-Alanine ((D)-Ala)

9

I

N-Methyl-leucine (MeLeu)

10

J

N-Methyl-leucine (MeLeu)

11

K

N-Methyl-valine (MeVal)

The relationship between the position numbering and the “Letter in Formula I” has been arbitrarily assigned for the purpose of defining the structure of the compounds of the present invention and does not represent any known convention for designating amino acids in cyclosporin analogues as such.

The compounds of the present invention are derived from known compounds of the cyclosporin family, including cyclosporin A, or other cyclosporins that have in the first amino acid position (according to cyclosporin nomenclature) of the cycloundecapeptide the MeBmt1, Deoxy-MeBmt1, Deoxy-Bmt1, or Bmt1. The novel compounds of the present invention all possess structurally modified amino acids at the one position. In addition to the modification to the amino acids at the one position, it is also within the scope of the present invention to include derivatives where structural changes have also been made simultaneously to one or more of the amino acids at positions two through eleven.

The present invention also provides novel chemical and biocatalytic processes for the preparation of novel cyclosporin derivatives. One such process involves the use of a biocatalyst for the conversion of members of the cyclosporin family, especially cyclosporin A, to novel cyclosporin derivatives that possess biological activity that make them useful as pharmaceutical compounds. This process involves the transformation of the MeBmt1, in cyclosporin A for example, to a new amino acid residue (4R)-4-((E)-2-keto-3-butenyl)-4, N-dimethyl-L-threonine (also systematically named (2S,3R,4R,5E)-3-hydroxy-4-methyl-2-(methylamino)-7-oxo-5-octenoic acid). The net effect of this biocatalytic process is to convert the amino acid side chain terminus from the “(E)-2-butenyl” moiety to a terminal “methyl vinyl ketone”, as shown below.

The novel cyclosporin methyl vinyl ketone (Cs-MVK, Formula III) derivatives possess biological activities that make them useful as pharmaceutical agents to treat a variety of medical conditions or disorders. The methyl vinyl ketone functional group also makes these compounds useful synthetic intermediates from which to make additional novel derivatives, as shown below.

Therefore, another aspect of the present invention relates to subjecting the compounds of Formula III to further chemical or biocatalytic manipulation, which leads to the production of novel compounds possessing pharmaceutical utility. Structural modifications produced by this iterative type of process are not restricted to the amino acid one position, but can take place on one or more of the other ten amino acids, positions two through eleven, around the cycloundecapeptide.

An alternative method for the preparation of the Cs-MVK derivatives is also disclosed, where a chemical oxidation that does not require the use of biocatalysts is performed to transform cyclosporins, including cyclosporin A, to the cyclosporin methyl vinyl ketones of Formula III.

It is well known that the amino acid at the one position (MeBmt1, Deoxy-MeBmt1 Deoxy-Bmt1 or Bmt1) of the cycloundecapeptide of cyclosporins, including cyclosporin A, plays a very important role in the biological activity of the cyclosporins. As a result of these structural changes to the amino acid in the one position, the novel cyclosporin derivatives of the present invention possess pharmaceutical utility towards several therapeutic indications.

The cyclosporins are best known for their immunosuppressive effects exerted by their selective action on T-lymphocytes of the immune system. Compounds disclosed in the present invention that possess inhibitory activity against calcineurin, an intracellular protein phosphatase involved in the regulation of intracellular lymphocyte (T-cell) signaling in the mammalian immune system, display immunosuppressive activity in mammals. The drug interaction with calcineurin occurs with a complex between cyclophilin A (the intracellular receptor for cyclosporins) and cyclosporin A that first forms. The major consequence of calcineurin inhibition is that dephosphorylation of the transcription factor nuclear factor of activated T-cells (NF-AT) does not occur and transcription of the cytokine interleukin-2 (IL-2) is inhibited. In in vitro and in vivo tests, the effect observed is the inhibition of T-cell proliferation and lymphocyte cell differentiation (T-lymphocytes to B-lymphocytes). Compounds disclosed in the present invention that have this biological activity profile are in the same class as cyclosporin A, and administration of these compounds suppresses the immune response in organ transplant patients and, thus, prevents allograft rejection. Compounds disclosed in the present invention in this class also possess utility in the treatment of autoimmune and chronic inflammatory diseases like asthma, rheumatoid arthritis, multiple sclerosis, psoriasis, and ulcerative colitis, to name only a few.

Another aspect of the present invention provides new cyclosporin A analogues that possess other useful biological activities that are dissociated from immunosuppressive activity. Some cyclosporin derivatives of the present invention retain good binding affinity toward cyclophilin A, but lack calcineurin inhibitory activity and, therefore, lack immunosuppressive activity. Cyclophilin A, like other cyclophilins, is a peptidyl-prolyl cis-trans isomerase (PPIase) which is important for protein folding or chaperone activities. Cyclosporin A and FK-506 have been shown to possess neurotrophic activity in mammals (Snyder et al., “Neural Actions of Immunophilin Ligands,” Trends in Pharmacological Sciences, 19:21-26 (1998), which is hereby incorporated in its entirety). It has also been reported that analogues of cyclosporin A and FK-506 that lack immunosuppressive activity but retain potent PPIase inhibitory activity retain neurotrophic activity. This demonstrates the feasibility of dissociating the immunosuppressive and neurotrophic activities. These compounds have been shown to possess the therapeutic utility for the treatment of a wide range of neurodegenerative diseases like diabetic neuropathy, amyotrophic lateral sclerosis, spinal cord injury, Alzheimer's disease, Parkinson's disease, and stroke, to name a few. Compounds disclosed in the present invention possess similar biological activity profiles and, therefore, utilities.

Other cyclosporin derivatives devoid of immunosuppressive activity have shown the ability to disrupt the human immunodeficiency virus (HIV) life cycle, e.g., SDZ NIM 811 (Mlynar et al., “The Non-immunosuppressive Cyclosporin A Analog SDZ NIM 811 Inhibits Cyclophilin A Incorporation Into Virions and Virus Replication in Human Immunodeficiency Virus Type 1 Infected Primary and Growth-Arrested T Cells,” J. Gen. Virol., 78(4):825-835 (1997), which is hereby incorporated by reference in its entirety). Binding to cyclophilin A is a prerequisite for HIV-1 inhibition by cyclosporins. Cyclophilin A was demonstrated to bind to HIV-1 p24gag and this cyclophilin-Gag interaction leads to the incorporation of cyclophilin A into HIV-1 virions. Compounds disclosed in the present invention that function in a manner like SDZ NIM 811 inhibit this protein interaction, which is likely to be the molecular basis for their antiviral activity.

While much of the biological activity described above requires cyclosporins, like cyclosporin A, to cross the cell plasma membrane and interact with intracellular protein targets like the cyclophilins and calcineurin, some biological activities result from the interaction of cyclosporins with proteins in the plasma membrane. Cyclosporin A is a broad and relatively unselective inhibitor of seven transmembrane-G-protein coupled receptors (7-TM-GPCR) and 12 transmembrane (TM) channels and transporters. One of these transporters is the multidrug resistance-1 P-glycoprotein (MDR1-encoded Pgp), a 12 TM ATP binding cassette (ABC) transporter. The cell specific expression of the MDR1 Pgp sustains house-keeping functions (e.g., at the blood-brain barrier), toxin exclusion (e.g., in the gut), and toxic metabolites clearance (e.g., in the liver), but MDR1 Pgp is also a flippase for selective membrane phospholipids (Loor, “Cyclosporins and Related Fungal Products in the Reversal of P-Glycoprotein-Mediated Multidrug Resistance,” In Multidrug Resistance in Cancer Cells, Gupta et al., eds. pp 387-412, John Wiley & Sons Ltd.: Chichester (1996), which is hereby incorporated by reference in its entirety). This Pgp activity also restricts anticancer drug accumulation by the cells, causing the MDR phenotype of some tumor cells. Several cyclosporin derivatives were found to behave as highly potent and selective inhibitors of drug transport by the MDR1 Pgp. One such cyclosporin derivative, known as Valspodar ([3′-keto-MeBmt1, Val2]-CsA), is more potent and selective than CsA (Loor, “Valspodar: Current Status and Perspectives,” Exp. Opin. Invest. Drugs, B 8:807-835 (1999), which is hereby incorporated by reference in its entirety). Several cyclosporin derivatives disclosed in the present invention, like Valspodar, lack immunosuppressive activity (and other collateral activities of CsA), and are useful for chemosensitization of MDR tumor cells.

The compounds disclosed in the present invention may be administered neat or with a pharmaceutical carrier to warm blooded mammals. Compounds disclosed in the present invention can be administered to patients for the treatment of immunoregulatory disorders, autoimmune disease, HIV infection, neurodegenerative disease, for the prevention of organ transplant rejection, and for the chemosensitization of tumor cells resistant to chemotherapy.

For treatment of the above mentioned diseases, therapeutically effective doses of cyclosporin compounds of the present invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral, as used herein, includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

The pharmaceutical compositions containing the active ingredient may be in the form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. The pharmaceutical compositions of the present invention contain the active ingredient formulated with one or more pharmaceutical excipients. As used herein, the term “pharmaceutical excipient” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of pharmaceutical excipients are sugars such as lactose, glucose, and sucrose; starches such as corn starch or potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; non-toxic, compatible lubricants such as sodium lauryl sulfate and magnesium stearate; as well as coloring agents, releasing agents, sweetening, and flavoring and perfuming agents. Preservatives and antioxidants, such as ethyl or n-propyl p-hydroxybenzoate, can also be included in the pharmaceutical compositions.

Dosage forms for topical or transdermal administration of compounds disclosed in the present invention include ointments, pastes, creams, lotions, gels, plasters, cataplasms, powders, solutions, sprays, inhalants, or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers, as may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

For nasal administration, compounds disclosed in the present invention can be administered, as suitable, in liquid or powdered form from a nasal applicator. Forms suitable for ophthalmic use will include lotions, tinctures, gels, ointment and ophthalmic inserts, as known in the art. For rectal administration (topical therapy of the colon), compounds of the present invention may be administered in suppository or enema form, in solution in particular, for example in vegetable oil or in an oily system for use as a retention enema.

Compounds disclosed in the present invention may be delivered to the lungs by the inhaled route either in nebulizer form or as a dry powder. The advantage of the inhaled route, over the systemic route, in the treatment of asthma and other diseases of airflow obstruction and/or chronic sinusitis, is that patients are exposed to very small quantities of the drug and the compound is delivered directly to the site of action.

Dosages of compounds of the present invention employed for the treatment of the maladies identified in the present invention will vary depending on the site of treatment, the particular condition to be treated, the severity of the condition, the subject to be treated (who may vary in body weight, age, general health, sex, and other factors) as well as the effect desired.

Dosage levels ranging from about 0.05 mg to about 50 mg per kilogram of body weight per day are useful for the treatment of the conditions or diseases identified in the present invention. This means the amount of the compound disclosed in the present invention that is administered will range from 2.5 mg to about 2.5 gm per patient per day.

The amount of active ingredient that may be combined with the pharmaceutical carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 2.5 mg to 2.5 gm of active compound of the present invention compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 5 mg to about 500 mg of active compound of the present invention. Dosage for topical preparation will, in general be less (one tenth to one hundredth) of the dose required for an oral preparation.

The Synthesis of Novel Cyclosporin A Derivatives

The starting materials for the preparation of novel cyclosporin derivatives of the present invention are members of the cyclosporin family, including cyclosporin A (CsA). Selective oxidation of the MeBmt1, deoxy-MeBmt1, Bmt1, or deoxy-Bmt1 side chain of a cyclosporin can be achieved biocatalytically by the use of enzymes from the laccase family.

Laccases (EC 1.10.3.2) are multi-copper oxidases that can catalyze the oxidation of a range of reducing substances with concomitant reduction of molecular oxygen (Xu et al, “Redox Chemistry in Laccase-Catalyzed Oxidation of N-Hydroxy Compounds,” Applied and Environmental Microbiology, 66:2052-2056 (2000), which is hereby incorporated by reference in its entirety). It has been shown that many compounds that would appear to possess comparable redox potentials are not laccase substrates due to unfavorable kinetics. Under certain conditions, however, these compounds can be indirectly oxidized by laccase through the mediation of small, redox active laccase substrates. Some known mediators of laccase catalysis are 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and N-hydroxy compounds such as 1-hydroxybenzotriazole (HOBT), violuric Acid (VA) and N-hydroxyacetanilide (NHA).

Laccase from Trametes versicolor in combination with mediators, such as ABTS, NHA, and HOBT, are used as bleaching agents for lignin degradation and pulp bleaching in the paper industry. In organic synthesis, laccase mediated oxidation is used for the transformation of an aromatic methyl group to an aromatic aldehyde (Fritz-Langhals et al., “Synthesis of Aromatic Aldehydes by Laccase-Mediator Assisted Oxidation,” Tetrahedron Lett., 39:5955-5956 (1998), which is hereby incorporated by reference in its entirety) as well as the conversion of a benzyl alcohol to benzaldehyde (Potthast et al., “A Novel Method for the Conversion of Benzyl Alcohols to Benzaldehydes by Laccase-Catalyzed Oxidation,” J. Mol. Cat., A 108:5-9 (1996), which is hereby incorporated by reference in its entirety).

The HOBT-mediated laccase oxidation of cyclosporins is a novel application for the laccase enzyme. Treatment of cyclosporins, including cyclosporin A, with HOBT-mediated laccase oxidation conditions results in the preparation of cyclosporin methyl vinyl ketones (Cs-MVK) of Formula III. The net effect of this biocatalytic process is to convert the position one amino acid side chain terminus from the “(E)-2-butenyl” moiety to a terminal “methyl vinyl ketone,” as shown in Scheme 1.

The more likely products that are expected oxidation are products of allylic oxidation of the methyl or methylene positions, i.e., primary or secondary alcohols and more highly oxidized products arising from these, i.e., aldehydes or ketones. These expected products, however, are minor reaction products at best. The formation of the Cs-MVK (Formula III) as the major product via the HOBT-mediated laccase oxidation is unexpected and unprecedented. This biocatalytic process works best using HOBT as the mediator, however the present invention includes the use of other known mediators like ABTS, VA, NHA, or other mediators known in the art. Also, the present invention includes the use of laccase enzyme from other known sources, e.g., Trametes villosa, Pleurotus ostreatus, Polyporus versicolor, or other known organisms from which laccase has been found.

The selective oxidation of the MeBmt1, deoxy-MeBmt1, Bmt1, or deoxy-Bmt1 side chains of cyclosporins to Cs-MVKs (compounds of Formula III) can also be practiced by chemical transformation that does not require the use of a biocatalyst such as the laccase enzyme. Punniyamurthy et al., “Cobalt Catalyzed Allylic and Benzylic Oxidations with Dioxygen in the Presence of Ethyl 2-Oxocyclopentanecarboxylate,” Tetrahedron Lett., 35:4003-4006 (1994), which is hereby incorporated by reference in its entirety, has reported this type of functional group transformation, where cis-2-octene is converted to 2-keto-oct-3-ene using a catalytic amount of cobalt (II) Schiff's base complex [bis(salicylidene-N-(methyl-3-hydroxypropionate))] and molecular oxygen, as a “surprising” result.

One embodiment of the present invention relates to a process for effecting allylic oxidation which utilizes an alkali metal periodate and an alkyl hydroperoxide (see U.S. Pat. No. 5,869,709 to Marwah et al., which is hereby incorporated by reference in its entirety). Cyclosporin A, when subjected to treatment with t-butyl peroxide and sodium periodate in acetone or methyl isobutyl ketone at room temperature or with gentle heating, results in the formation of the CsA MVK (IIIa) product, as shown in Scheme 2.

While the periodate/peroxide conditions described above are effective for reaction scales of a gram or less of cyclosporin starting materials, the yield of the Cs-MVK product are lower when the reaction is run on larger scale with significant amounts of unreacted cyclosporine isolated. Further optimization of the periodate/peroxide conditions involves the use of t-butyl hydroperoxide (50-100 equivalents), potassium periodate (5-10 equivalents), and a crown ether (18-crown-6, 4-10 equivalents) in an acetone-benzene-water (1.0:1.0:1.5) solvent mixture at room temperature for three days. The oxidation of cyclosporins proceed under these novel conditions on a multigram scale in nearly complete conversion of the cyclosporin starting materials to Cs-MVK products in good yields.

Another embodiment of the present invention relates to an oxidation condition for allylic groups that utilizes catalytic N-hydroxydicarboxylic acid imides, e.g., N-hydroxyphthalimide, dibenzoyl peroxide, refluxing acetone or isobutyl methyl ketone and air (see U.S. Pat. No. 5,030,739 to Foricher et al., which is hereby incorporated by reference in its entirety). These conditions are appealing because of the use of a similar N-hydroxy mediator used in the biocatalytic process. When cyclosporin A is subjected to these conditions, the desired product, CsA-MVK (IIIa), is detected by proton NMR analysis of the reaction mixture, but the major product isolated is an N-hydroxyphthalimide CsA adduct. Upon resubjecting this adduct to the reaction conditions, CsA MVK is isolated as the major product. It is important to note that Cs-MVK products isolated by either of the chemical methods described in the present invention would not be predicted based on the products reported in the previously cited patents.

Further chemical modification of Cs-MVK products of the present invention can be performed by sodium borohydride reduction of the ketone to give a diastereomeric mixture (1:1) of cyclosporin alcohols, referred to as cyclosporin alcohols A (IVa-A) and B (IVa-B) shown in Scheme 3. The cyclosporin alcohols (IVa-A & IVa-B) are separable by semi-preparative reverse phase HPLC (C8 column). Cyclosporin alcohol “isomer A” when subjected to treatment with acetic anhydride, DMAP and pyridine in dichloromethane gives a mixture of cyclosporin alcohol monoacetyl ester 1, cyclosporin alcohol monoacetyl ester 2 and cyclosporin alcohol diacetate of IVa-A. The products are purified and separated by semi-preparative reverse phase HPLC (C8 column). Selective acylation of CsA alcohol “isomer B” is performed by enzymatic acylation with a lipase in an organic solvent. The lipase can be from Pseudomonas cepacia or Pseudomonas fluorescens, and can be a native lipase or a genetically modified lipase. In another embodiment, the lipase can be immobilized to a solid support. Examples of the organic solvent are methyl-tert-butyl ether, toluene, pyridine, or mixtures thereof, and mixtures with N,N-dimethyl formamide. When the CsA alcohol “isomer B” (IVa-B) is stirred in methyl tert-butyl ether with vinyl butyrate and immobilized lipases AH and AK, the monobutyrate ester of IVa-B is prepared.

Other cyclosporin derivatives disclosed in the present invention can be prepared by applying biocatalytic or chemical methods in an iterative process. Scheme 4 shows CsA-MVK (IIIa), when incubated with Saccharopolyspora hirsute subspecie hirsuta (Microbe no. 27875-ATCC), leading to the formation of γ-hydroxy-MeLeu4 CsA-MVK (see Example 12). It is also possible to reverse the order of biocatalytic reactions by first modifying cyclosporin A, e.g., converting CsA to [γ-hydroxy-MeLeu9]CsA by incubation of CsA with Streptomyces catenulae (Microbe no. 23893-ATCC) (see Example 13). Then, upon isolation and purification by reversed phase semi-preparative (C8) chromatography, [γ-hydroxy-MeLeu9]CsA is subjected to HOBT-mediated laccase c oxidation to produce [γ-hydroxy-MeLeu9] CsA-MVK (see Example 14).

In addition, the cyclosporin methyl vinyl ketone of Formula III can be chemically modified by oxidative cleavage of the double bond to give the aldehyde of Formula V. This transformation can be performed directly from the cyclosporin methyl vinyl ketone of Formula III or the cyclosporin alcohol of Formula IV. Ozonolysis of either compound followed by reductive workup (zinc/acetic acid or dimethyl sulfide) provides the cyclosporin aldehyde (Va) in good yield as shown in Scheme 5.

The cyclosporin aldehyde of Formula V provides another useful 15 synthetic intermediate from which to prepare novel cyclosporin derivatives of the present invention. Phosporous ylide chemistry, i.e., the Wittig reaction and Wittig-Homer-Emmons reaction, can be successfully performed on the cyclosporin aldehyde, as shown in Scheme 6. This chemistry converts the aldehyde to a substituted olefin of Formula VI, thus extending the carbon chain and introducing a variety of novel substituents attached to the olefin.

There is an abundance of phosphorous ylide precursors available commercially or readily prepared by following procedures found in the chemistry literature. Several representatives of this class of reagents that are utilized in the present invention are as follows:

Iodomethyltriphenyphosphonium iodide;

Methyltriphenylphosphonium bromide;

[3-(Dimethylamino)propyl]triphenylphosphonium bromide;

n-Propyltriphenylphosphonium bromide;

(3,3-Dimethylallyl)triphenylphosphonium bromide;

Methyl diethylphosphonoacetate;

Diethyl cyanomethylphosphonate;

Trans-2-butenyltriphenylphosphonium bromide,
to name only a few. The above list is not intended to limit the scope of the present invention to the use of these reagents only.

The reactive ylide species in the Wittig or Wittig-Homer-Emmons reaction are typically generated by treatment of the above phosphonium salts or phosphonates with a strong base. Examples of bases that can be used in the present invention include sodium hydride or sodium bis(trimethylsilyl)amide. Typically, a large excess (5 to 15 equivalents) of the phosphorous ylide is used to react with compounds of Formula V. Reaction temperatures are often maintained between −78° C. and 0° C. The compounds of Formula VI isolated from this reaction may exist as cis and trans isomers of the alkene.

Examples of compounds of Formula VI include the following compound:

as well as other compounds where the position one amino acids are of the following formulas:

Another aspect of the present invention relates to the transformation of cyclosporin aldehyde of Formula V into the corresponding carboxylic acid and its derivatives, such as carboxylic esters and amides. Treatment of cyclosporin aldehyde (Va) with tert-butyl hypochlorite, followed by addition of methanol and pyridine produces the cylosporin ester (Scheme 7).

Reduction of cyclosporine aldyhyde (Va) with sodium borohydride provides cyclosporine diol in quantative yield (Scheme 8). Further chemical modification of the diol can be performed by selective esterfication on the primary alcohol with various acid chlorides or acid anhydrides in the presence of pyridine to afford mono-esters of Formula VII.

Examples of compounds of Formula VII include the following compounds:

Another aspect of the present invention relates to the preparation of cyclosporine amines and amine derivatives from cyclosporine aldehyde (Va). Reductive amination of CsA aldehyde (Va) with ammonium acetate and sodium cyanoborohydride in the presence of acetic acid in methanol generates cyclosporine amine (R9═H), while treatment of aldehyde (Va) with methylamine followed by reduction with sodium borohydride produces cyclosporine methylamine (R9═CH3), as shown in Scheme 9. Further modifications of cyclosporin amine or cyclosporine methylamine include alkylation or acylation to generate amine derivatives of Formula VIII. The alkylation can be accomplished by reacting the cyclosporin amine or cyclosporine methylamine in the presence of an alkyl halide such as alkyl bromide, alkyl chloride, or alkyl iodide. Alternatively, the acylation can be accomplished by reacting the cyclosporin amine or cyclosporine methylamine in the presence of an acid anhydride or acid chloride, or in the presence of a sulfonic acid anhydride or sulfonyl chloride.

Examples of compounds of Formula VIII include the following compounds:

Another aspect of the present invention relates to the preparation of novel cyclosporine vinyl halides via Wittig reaction or Takai reaction (Scheme 10). Wittig reaction of CsA aldehyde (Va) with phosphorous ylide that can be generated from iodomethyltriphenylphosphonium iodide and sodium bis(trimethylsilyl)amide affords cis-isomer of cyclosporine vinyl iodide. The trans-isomer of cyclosporine vinyl iodide can be prepared by treatment of the aldehyde (Va) with iodoform in the presence of chromium(II) chloride. Other cyclosporine vinyl halides, such as vinyl bromide and vinyl chloride, can be also prepared using the methods outlined in Scheme 10.

Further chemical modification on cyclosporine vinyl iodide includes palladium mediated coupling with organotin or organozinc reagents to provide novel cyclosporine olefin of Formula VI, as shown in Scheme 11.

Examples of compounds of Formula VI include the following compounds:

Another aspect of the present invention relates to the preparation of the novel cyclosporine diol of Formula IX by treatment of cyclosporine aldehyde of Formula V with various Grignard reagents at −78° C. or organozinc reagents at 0° C. (Scheme 12). Many organozinc reagents are commercially available or can be generated from the corresponding Grignard reagents and zinc chloride.

Examples of compounds of Formula IX include the following compounds:

The present invention also discloses compounds prepared from cyclosporin A aldehyde (Va), where the position one amino acid is an amino acid of the following formula:

Other examples of compounds prepared from cyclosporin A aldehyde (Va) include the following compounds:

Another aspect of the present invention relates to the preparation of novel cyclosporine diene analogues of Formula X. Dehydration of cyclosporine alcohol of Formula IV with Burgess reagent at 60-80° C. in benzene affords cyclosporine diene (X═OAc), as shown in Scheme 13. The acetyl protecting group can be removed by treatment with potassium carbonate in methanol to give cyclosporine diene (X═OH). Olefin metathesis of cyclosporine diene with various olefin species in the presence of Grubbs' catalyst provides substituted diene of Formula X smoothly. It is interesting that olefin metathesis only occurs on outside carbon-carbon double bond moiety, while the inside carbon-carbon double bond remains unchanged.

Examples of compounds of Formula X include the following compounds:

Another aspect of the present invention relates to the preparation of novel cyclosporine methyl ketones of Formula XI. Conversion of cyclosporine methyl vinyl ketone of Formula III to cyclosporine methyl ketone of Formula X can be conducted under hydrogenation with palladium on carbon, as shown in Scheme 14.

The cyclosporine methyl ketone of Formula XI is another useful synthetic intermediate which can be converted to novel cyclosporine analogues of the present invention. Wittig reaction of cyclosporine methyl ketone of Formula XI with various phosphorous ylide species provides novel olefin of Formula XII (Scheme 15). Cyclosporine olefin of Formula XII can be prepared via an alternative synthetic passway, as shown in Scheme 15. Treatment of cyclosporine methyl ketone of Formula XI with Grignard reagents or organozinc reagents affords novel alcohol of Formula XIII smoothly. Dehydration of alcohol of Formula XIII with Burgess reagent generates cyclosporine olefin of Formula XII.

Examples of compounds of Formula XIII include the following compounds:

Examples of compounds of Formula XII include the following compounds:

The present invention also discloses compounds prepared from cyclosporin A methyl ketone of Formula X, where the position one amino acid is an amino acid of the following formula:

Other examples of compounds prepared from cyclosporin A methyl ketone of Formula X include the following compounds:

Another aspect of the present invention relates to the alternative method of preparing the novel cyclosporin olefins of Formula VI from cyclosporine aldehyde of Formula XIV. Transformation of cyclosporin A (X═OAc) into cyclosporine aldehyde of Formula XIV (X═OAc) can be performed under standard ozonation conditions. Treatment of aldehyde of Formula XIV with Grignard reagents at −78° C. or organozinc reagents at 0° C. provides alcohol of Formula XV in good yield. Organozinc reagents used in this reaction can be either commercially available or generated from the corresponding Grignard reagent and zinc chloride. Dehydration of alcohol of Formula XV with Burgess reagent in benzene affords olefin of Formula VI smoothly (Scheme 16).

Examples of compounds of Formula XV include the following compounds:

Examples of compounds of Formula VI include the following compounds:

The carbon-carbon double bond in novel cyclosporine olefin of Formula VI can be reduced by hydronation with palladium on carbon to afford novel closporine analogues of Formula XVI, as shown in Scheme 17.

Examples of compounds of Formula XVI include the following compounds:

EXAMPLES

The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.

Example 1Materials and Methods (for Examples 3-14)

Reagents were purchased from commercial sources and used as received. Solvents for reactions and isolations were of reagent or spectrophotometric grade and used without further treatment. Anhydrous tert-butyl methyl ether was maintained dry over molecular sieves, previously activated at 105° C. for at least 12 h. Solvents were removed under vacuum with either a Buchi Rotavapor R-114 with Waterbath B-480, GeneVac HT-12 Atlas Evaporator with Vapour Condenser VC3000D and CVP 100 pump, or a Savant SpeedVac® Plus SC210A with VaporTrap RVT4104. 1H and 13C-NMR spectra were collected in CDCl3, on a Bruker WM-360 spectrometer, with signals reported in ppm and internally referenced to TMS (δ 0.0), or CDCl3 (δ 77.23). Centrifugation was accomplished using either a Beckman J2-MC Centrifuge, Fisher Scientific Marathon 26KM Centrifuge or Eppendorf Centrifuge 5810. Microbial cultures were shaken on a New Brunswick Innova 5000 rotary shaker inside a thermostatically controlled room maintained at 29° C. Aseptic transfer and inoculation techniques were performed inside a Nuaire NU-425-400 biological safety cabinet. Semi-preparative reversed-phase HPLC purifications were performed on a Gilson system with model 306 pumps, model 215 liquid handler, and a Perkin-Elmer LC Oven 101 column heater, employing a Zorbax StableBond Rx-C8 column, 21.2×250 mm, 7 μm packing. Analytical HPLC was performed on a Shimadzu system with SCL-10A system controller, SPD-M10A diode array detector, SIL-10AD auto injector, LC-10AT liquid chromatograph, DGU-14A degasser, and CTO-10A column oven, employing a Zorbax StableBond Rx-C8 column, 4.6×150 mm, 3 μm packing. LC/MS analysis was performed using a Perkin-Elmer SciEx API 2000 LC/MS/MS system with a Perkin Elmer Series 2000 Micropump and Zorbax StableBond Rx-C8 columns, 4.6×50 mm, 3 μm packing, at 70° C.

Example 2Culture Growth and Maintenance

Cultures were maintained on agar slants stored at 4° C. or as suspensions in 10% glycerol at −85° C. Mycelium and/or spores from slants were used to inoculate 125 mL DeLong flasks containing 12.5 mL soybean flour-glucose growth medium. Stage I cultures were shaken at 250 rpm for 48-72 h. A 10% inoculum was transferred from Stage I culture to 125 mL DeLong flasks containing 12.5 mL soybean flour-glucose medium to start Stage II cultures. Stage II cultures were grown at 250 rpm for 24 hours before being dosed with cyclosporin-type molecules. Cultures from cryo-preserved vials were initiated by aseptically transferring the contents of one vial (appropriately warmed to room temperature beforehand) to a 125 mL DeLong flask to start Stage II cultures. The growth medium was prepared in two parts. Part A consisted of soybean flour (1%), yeast extract (1%), NaCl (1%) and K2HPO4 (1%) in deionized water. The pH was adjusted to 7 with 50% HCl. Part B consisted of a 4% glucose solution in deionized water. Parts A and B were autoclaved separately at 121° C. and 15 psi for 20 min, mixed together under a sterile environment and allowed to cool to room temperature prior to use.

Example 3Preparation of Cyclosporin A Methyl Vinyl Ketone (IIIa) by a Biocatalytic Method

Example 4Preparation of Cyclosporin A Methyl Vinyl Ketone (IIIa) by a Chemical Method Using N-Hydroxypthalimide and Benzoyl Peroxide

A solution of N-hydroxypthalimide (68 mg, 0.41 mmoles) and CsA (500 mg, 0.41 mmoles) in isobutyl methyl ketone (5.0 mL) was heated at 54° C. The solution was treated with dibenzoyl peroxide (75% in water) (80 mg, 0.27 mmoles) and subsequently; air was bubbled through the reaction mixture while stirring vigorously for 15 h. When consumption of CsA was complete as determined by TLC (3% methanol in chloroform), the reaction was concentrated to an oily residue. The latter was diluted with carbon tetrachloride and the reaction mixture was stirred for 1 h at 40° C. The excess N-hydroxypthalimide was filtered and the filtrate was concentrated to dryness under reduced pressure. The resulting oily residue was purified by column chromatography eluting with 2% methanol in chloroform. The main fraction was concentrated to give 380 mg of a white solid. 1H NMR and MS indicated the presence of a new product that was probably the cyclosporin A methyl vinyl ketone-N-hydroxyphthalimide adduct. A small amount of this product (100 mg) was subjected once again to the same protocol. A new spot was observed by TLC, which upon purification by column chromatography (2% methanol in chloroform on silica gel) gave the product CsA-MVK (25 mg) that matched by in all respects the CsA-MVK isolated from the biocatalysis method.

Example 5Preparation of Cyclosporin A Methyl Vinyl Ketone(IIIa) by a Chemical Method Using tert-Butyl Hydroperoxide and Sodium Periodate

To a mixture of CsA (400 mg, 0.33 mmoles) in isobutyl methyl ketone (5.0 mL) was added tert-butyl hydroperoxide (70% aqueous solution, 2.5 mL). Upon addition of sodium periodate (425 mg, 1.99 mmoles) at room temperature, the resulting mixture was stirred vigourously at 50° C. for 72 h. The mixture was diluted with dichloromethane (10 mL) and the organic layer was separated, washed thoroughly with water and then stirred with an aqueous sodium sulfite solution (15% aqueous solution, 30 mL) for 2 h. The organic layer was separated, dried over sodium sulfate and concentrated to dryness. The resulting residue was purified on silica gel eluting with 10% methanol in dichloromethane. The product isolated (370 mg) showed 70% of desired product as determined by 1HNMR along with unreacted cyclosporin A.

Unless otherwise noted, reagents and solvents were used as received from commercial suppliers. Proton and 19F nuclear magnetic resonance spectra were obtained on a Bruker AC 300 or a Bruker AV 300 spectrometer at 300 MHz for proton and 282 MHz for fluorine, or on a Bruker AMX 500 spectrometer at 500 MHz for proton. Spectra are given in ppm (δ) and coupling constants, J, are reported in Hertz. Tetramethylsilane was used as an internal standard for proton spectra and the solvent peak was used as the reference peak for carbon spectra. Mass spectra were obtained on a Perkin Elmer Sciex 100 atmospheric pressure ionization (APCI) mass spectrometer, or a Finnigan LCQ Duo LCMS ion trap electrospray ionization (ESI) mass spectrometer. HPLC analyses were obtained using a Dynamax C18 column (200×4.5 mm) or Luna C18(2) column (250×4.6 mm) with UV detection at 210 nm using a standard solvent gradient program (Method A; Table 2) and oven temperature at 65° C. Semi-prepare HPLC were performed using a Dynamax C18 column (60 A, 8 um) or a Luna C18(2) column (250×21.2 mm) with a standard solvent gradient program (Method B; Table 3) and oven temperature at 70° C. Elemental analyses were performed by Quantitative Technologies, Inc. (Whitehouse, N.J.).

A solution of CsA aldehyde (XIV, 400 mg, 0.32 mmol) in THF (10 mL) was cooled to −78° C. and treated with 4-biphenylmagnesium bromide and allowed to stir for 30 min under N2 atmosphere. Reaction was quenched with saturated ammonium chloride solution at −78° C., extracted with ethyl acetate, washed with brine, dried over sodium sulfate, and concentrated in vacuo. The crude product was purified by washing through a short silica gel column (9:1 hexanes/ethyl acetate to 9:1 ethyl acetate/methanol) to afford the crude alcohol (415 mg, 92%) as a pale yellow solid.

Vinylmagnesium bromide (1.0 M in THF, 1.0 mL, 1.0 mmol) was added in 4 portions to a solution of CsA aldehyde (XIV, 300 mg, 0.24 mmol) in THF (10 mL) at −78° C. under nitrogen in 1 h. After addition the resulted mixture was stirred at −78° C. for 15 min., and then was quenched with saturated aqueous NH4Cl solution (2 mL) at −78° C. The mixture was allowed to warm up to room temperature, and then poured in 10 mL of saturated aqueous NH4Cl solution, extracted with EtOAc (3×25 mL). The combined organic layers were washed with saturated aqueous H4Cl solution and brine, dried over NaSO4. Concentrated to dryness to give 300 mg of white solid. The crude alcohol was used for next step without further purification.

A mixture of CsA alcohol from Example 79 (100 mg, 0.077 mmol), Burgess reagent (27 mg, 0.115 mmol) and benzene (4 mL) was heated at reflux for 1 h, and then cooled to room temperature, diluted with ether, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by semi-preparative HPLC to afford the cis-isomer (10 mg, 10%) as a white solid: ESI MS m/z 1281 [C67H113N11O13+H]+.

Cyclosporin A and cyclosporin derivatives of the present invention were tested for biological activity in the mixed lymphocyte reaction (MLR) assay. The MLR assay was designed to measure 3H-thymidine uptake by human lymphocytes or murine splenocytes that are undergoing cell proliferation in an immune response to allogeneic stimulation. The murine system uses the H2 disparate inbred mouse strains: Balb/c (H2d) and C57B1/6 (H2b). The results of testing cyclosporin A and cyclosporin derivatives of the present invention in human and murine MLR were comparable. The MLR assay is useful for identifying CsA derivatives with immunosuppressive activity and to quantify this activity relative to the immunosuppressive activity of CsA.

For the purposes of testing compounds of the present invention, a one-way MLR was performed. In this method, the splenocytes of the C57B1/6 mice are γ-irradiated so as to act as stimulators of an immune response from the splenocytes from the Balb/c mice. First, spleens from Balb/C and C57B1/6 mice were surgically removed. Next, splenocytes were isolated by meshing each spleen and suspending with RPMI/HEPES/0.01% human serum albumin. Then, C57B1/6 splenocyte cells (stimulators) were γ-irradiated at 2000 rads. Cells were washed after irradiation. Next, stimulator and responder cells were counted at 1:20 dilution in Trypan. Cell populations were established at 5.12×106 cells per mL. Then, samples were plated in 96 well sterile tissue culture plates. To each well was added an aliquot (100 μL) of splenocytes (responders) from Balb/c mice and an aliquot (100 μL) of γ-irradiated splenocytes (stimulators) from C57B1/6 mice (final volume=200 μL with cell population of 2.5×105 cells). Aliquots of a 4 μg/mL stock solution of cyclosporin A and cyclosporin derivatives were measured and combined with the amount of media that resulted in 200 μL final volume. Concentrations of cyclosporin A in test wells tested were: 10.0, 20.0, 30.0, 40.0, and 60.0 ng/mL. Cyclosporin derivatives of the present invention were initially tested at 10, 100, and 1000 ng/mL drug concentrations to determine the range of potency and then retested at tighter concentration intervals to determine IC50 values (the inhibitory concentration of test compound determined to inhibit proliferation by 50% relative to control). To measure the effect of drug on proliferation, the plate was incubated for 3 days at 37° C. in 5% CO2. On day 4, 1 μCi/well of 3H-Thymidine was added and the plate was incubated for 24 hours. On day 5, cells were harvested onto a glass microfiber filtermat using a cell harvester. Dried filtermat and scintillation fluid were placed into sample bag and sealed. Then, the amount of radioactivity incorporated in the splenocytes was measured using a beta counter for 1 minute. Finally, averages and standard deviations for each drug were calculated and results were expressed as:
% Inhibition (% control)=(1−[average CPM of test drug÷average CPM of 0 drug])×100;
% Proliferation=100−% Inhibition

Initial screens were done at a fixed value of 100 ng/ml test compound. IC50s are calculated from 7 point concentration-response curves using GraphPad software. IC50 values for cyclosporin A, which was routinely run as the positive control in this immunosuppression assay, fell between 8-35 ng/ml. IC50 values for compounds of the present invention tested in this immunosuppression assay typically fall in the range: 100 ng/ml≦IC50≦1000 ng/ml. Compound IIIa of the present invention (synthesized according to methods described in Examples 3, 4, 5, and 6) gave an IC50 value of 310 ng/ml (cyclosporin A IC50=14 ng/ml) in this assay. The compound exemplified in Example 109 gave an IC50 value of 160 ng/ml (cyclosporin A IC50=16 ng/ml).

An alternative assay that was used to determine immunosuppression activity was the concanavalin A-stimulated splenocyte assay. In this assay, male BALB/c mice, at 5 to 7 weeks of age, are sacrificed by CO2 inhalation. Spleens are removed and dissociated by pushing through a nylon cell strainer. The splenocytes are washed in RPMI 1640/5% fetal calf serum (FCS) and pelleted at 400g. Red blood cells are then lysed by resuspending the cell pellet in ACK lysis buffer (150 mM NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, 3 ml per spleen) for 10 min at room temperature. After pelleting at 400 g, the cells are washed by resuspending in RPMI 1640/5% FCS and repelleting. The cell pellet is resuspended in RPMI 1640/5% FCS and again passed through a cell strainer to remove cell aggregates. The cells are then counted and adjusted to 2×106 cells/ml in RPMI 1640/10% FCS/50 μM 2-mercaptoethanol. Cell viability is assessed by Trypan blue staining. Cyclosporin A or test compound and two micrograms of concanavalin A are added to the wells of a 96 well plate prior to the addition of 2×105 splenocytes. The cells are cultured in a 37° C. CO2 incubator for 2 days and then pulsed with 1 μCi of [3H]-thymidine for 6 hours. Cells are harvested onto filtermats with a TomTec 96 well plate harvester and lysed with H2O. The filtermat and scintillation fluid are sealed in a plastic sleeve. [3H]thymidine incorporation is measured with a Wallac Trilux plate counter. Initial screens are done at a fixed value of 100 ng/ml test compound. IC50s are calculated from 7 point concentration-response curves using GraphPad software. IC50 values for this immunosuppressive assay were consistent with those determined in the previous method.

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.